Many natural biological molecules and their analogues, including proteins and polynucleotides, foreign substances and drugs, which are capable of influencing cell function at the sub-cellular or molecular level are preferably incorporated within the cell in order to produce their effect. For these agents the cell membrane presents a selective barrier which is impermeable to them. The complex composition of the cell membrane comprises phospholipids, glycolipids, and cholesterol, as well as intrinsic and exrinsic proteins, and its functions are influenced by cytoplasmic components which include Ca++ and other metal ions, anions, ATP, microfilaments, microtubules, enzymes, and Ca++-binding proteins, also by the extracellular glycocalyx (proteoglycans, glycose aminoglycans and glycoproteins). Interactions among structural and cytoplasmic cell components and their response to external signals make up transport processes responsible for the membrane selectivity exhibited within and among cell types.
Successful delivery of agents not naturally taken up by cells into cells has also been investigated. The membrane barrier can be overcome by associating agents in complexes with lipid formulations closely resembling the lipid composition of natural cell membranes. These formulations may fuse with the cell membranes on contact, or what is more common, taken up by pynocytosis, endocytosis and/or phagocytosis. In all these processes, the associated substances are delivered in to the cells.
Lipid complexes can facilitate intracellular transfers also by overcoming charge repulsions between the cell surface, which in most cases is negatively charged. The lipids of the formulations comprise an amphipathic lipid, such as the phospholipids of cell membranes, and form various layers or aggregates such as micelles or hollow lipid vesicles (liposomes), in aqueous systems. The liposomes can be used to entrap the substance to be delivered within the liposomes; in other applications, the drug molecule of interest can be incorporated into the lipid vesicle as an intrinsic membrane component, rather than entrapped into the hollow aqueous interior, or electrostatically attached to aggregate surface. However, most phospholipids used are either zwiterionic (neutral) or negatively charged.
An advance in the area of intracellular delivery was the discovery that a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), in the form of liposomes, or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes which are capable of adsorbing to cell membranes and being taken up by the cells either by fusion or more probably by adsorptive endocytosis, resulting in expression of the transgene [Felgner, P. L. et al. Proc. Natl. Acad. Sci., USA 84:7413-7417 (1987) and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.]. Others have successfully used a DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in combination with a phospholipid to form DNA-complexing vesicles. The Lipofectin™ reagent (Bethesda Research Laboratories, Gaithersburg, Md.), an effective agent for the delivery of highly anionic polynucleotides into living tissue culture cells, comprises positively charged liposomes composed of positively charged lipid DOTMA and a neutral lipid dioleyl phosphatidyl ethanol amine (DOPE) referred to as helper lipids. These liposomes interact spontaneously with negatively charged nucleic acids to form complexes, referred to as lipoplexes. When excess of positively charged liposomes over DNA negative charges are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces or introduced into the cells either by adsorptive endocytosis or fuse with the plasma membrane, both processes deliver functional polynucleotide into, for example, tissue culture cells. DOTMA and DOTAP are good examples for monocationic lipids. [Illis et al. 2001, ibid.]
Multivalent cations by themselves (including polyamines, inorganic salts and complexes and dehydrating solvents) have also been shown to facilitate delivery of macromolecules into cells. In particular, multivalent cations provoke the collapse of oligo and polyanions (nucleic acids molecules, amino acid molecules and the like) to compact structural forms, and facilitate the packaging of these polyanions into viruses, their incorporation into liposomes, transfer into cells etc. [Thomas T. J. et al. Biochemistry 38:3821-3830 (1999)]. The smallest natural polycations able to compact DNA are the polyamines spermidine and spermine. By attaching a hydrophobic anchor to these molecules via a linker, a new class of transfection vectors, the polycationic lipopolymers, has been developed.
Cationic lipids and cationic polymers interact electrostatically with the anionic groups of DNA (or of any other polyanionic macromolecule) forming DNA-lipid complexes (lipoplexes) or DNA-polycation complexes (polyplexes). The formation of the complex is associated with the release of counterions of the lipids or polymer, which is the thermodynamic driving force for lipoplex and polyplex spontaneous formation. The cationic lipids can be divided into four classes: (i) quaternary ammonium salt lipids (e.g. DOTMA (Lipofectin™) and DOTAP) and phosphonium/arsonium congeners; (ii) lipopolyamines; (iii) cationic lipids bearing both quaternary ammonium and polyamine moieties and (iv) amidinium, guanidinium and heterocyclic salt lipids.